Systems and methods for modeling veins and associated blood vessel components

ABSTRACT

A venous valve model includes a first layer having a central axis and a fluid channel extending between a fluid inlet and a fluid outlet formed in the first layer, wherein the fluid channel is defined by a pair of channel walls, a first venous valve formed in the first layer and positioned along the fluid channel, and a pair of first actuation chambers positioned adjacent the channel walls of the fluid channel, wherein the pair of first actuation chambers are configured to decrease a width of the fluid channel in response to pressurization of the pair of first actuation chambers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent applicationSer. No. 62/800,163 filed Feb. 1, 2019, and entitled “Systems andMethods for Modeling Veins and Associated Blood Vessel Components,”which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND

The structural, functional and environmental complexity of the venousblood vessels poses certain technical challenges for in vitroinvestigation of its physiology and pathology using traditional cellculture models. As a result, most research in this area has relied onexpensive and time-consuming ex vivo or in vivo animal studies that canoften fail to model biological responses in humans. These drawbacks ofexisting models can limit the understanding and the development of newtherapeutic approaches to diseases of the vein such as deep veinthrombosis.

For example, venous thrombi or blood clots may form at the sites ofvenous valves, the venous thrombi comprising a unique anatomy and wherethe behavior of blood flow venous thrombi may be extremely complex.Particularly, deep vein thrombosis (DVT) is a serious debilitatingcondition, often killing patients within thirty days of its onset. Thevenous thrombi originate inside venous valves and are generallyregulated through vascular activation and shape, unique blood flowand/or abnormal blood chemistry—three factors known as Virchow's triad.However, existing models cannot predict the regulation of blood clotsdue to the non-involvement of shape of the valves, flow and thecomposition of cells within the existing models. Additionally, animalmodels cannot provide a dissectible analysis of the Virchow's triad andmay often lead to poor predictions of mechanisms of DVT and drugs.

BRIEF SUMMARY OF THE DISCLOSURE

An embodiment of a venous valve model comprises a first layer having acentral axis and a fluid channel extending between a fluid inlet and afluid outlet formed in the first layer, wherein the fluid channel isdefined by a pair of channel walls, a first venous valve formed in thefirst layer and positioned along the fluid channel, and a pair of firstactuation chambers positioned adjacent the channel walls of the fluidchannel, wherein the pair of first actuation chambers are configured todecrease a width of the fluid channel in response to pressurization ofthe pair of first actuation chambers. In some embodiments, the firstlayer is formed from a three-dimensionally printed material. In someembodiments, the venous valve model comprises a pump in fluidcommunication with at least one of the pair of first actuation chambers,wherein the pump comprises an infusion mode configured to increase apressure within the at least one of the pair of first actuation chambersto decrease the width of the fluid channel and a withdraw modeconfigured to decrease a pressure within the at least one of the pair offirst actuation chambers to increase the width of the fluid channel. Incertain embodiments, the pump comprises a syringe pump. In certainembodiments, the first venous valve comprises a pair of leafletsdefining a pair of cusps of the first venous valve, and a flow channelpositioned between the leaflets. In some embodiments, the pair of firstactuation chambers are configured to decrease a width of the flowchannel of the first venous valve in response to the pressurization ofthe pair of first actuation chambers. In some embodiments, the pair offirst actuation chambers are positioned adjacent a first section of thefluid channel, the first layer further comprises a pair of secondactuation chambers positioned adjacent a second section of the fluidchannel located between the first section and the fluid outlet, andwherein the first venous valve is positioned between the first sectionand the second section, and the leaflets of the first venous valve areconfigured to direct fluid within the second section of the fluidchannel into the cusps of the first venous valve in response topressurization of the pair of second actuation chambers. In certainembodiments, the venous valve model comprises a pair of third actuationchambers positioned adjacent a third section of the fluid channellocated between the second section and the fluid outlet, a second venousvalve positioned between the second section and the third section, and apumping system comprising a plurality of pumps and configured tosimultaneously pressurize the first section and the third section of thefluid channel and depressurize the second section of the fluid channel.In certain embodiments, the first layer comprises a pair of chamberwalls positioned between the first pair of actuation chambers and thesecond pair of actuation chambers, wherein the pair of chamber wallsrestrict fluid communication between the first pair of actuationchambers and the second pair of actuation chambers. In certainembodiments, the venous valve model comprises a second layer comprisinga first air channel extending parallel with the fluid channel of thefirst layer, and a third layer comprising a second air channel extendingparallel with the fluid channel of the first layer, wherein the firstair channel, the second air channel, and the fluid channel are eachintersected by a plane extending orthogonally from the central axis.

An embodiment of a microfluidic chip for modelling flow through a veincomprises a body comprising a microchannel extending between a fluidinlet and a fluid outlet, wherein at least a portion of the microchannelis coated with endothelial cells that form vascular lumen, and a venousvalve formed in the body and positioned along the microchannel, whereinthe venous valve comprises a pair of leaflets defining a pair of cuspsof the venous valve, and a flow channel positioned between the leaflets.In some embodiments, the endothelial cells comprise human umbilical veinendothelial cells (HUVECs). In some embodiments, the HUVECs are coatedover a layer of an extracellular matrix (ECM). In some embodiments, thevascular lumen is treated with tumor necrosis-factor alpha (TNF-α) at adosage of less than 300 nanograms per milliliter (ng/ml). In certainembodiments, at least a portion of the pair of cusps is coated with theendothelial cells that form the vascular lumen. In certain embodiments,a width of the flow channel of the venous valve is between 25micrometers (μm) and 200 μm. In some embodiments, the body is formedfrom Polydimethylsiloxane (PDMS).

An embodiment of a method of forming a microfluidic chip for modellingflow through a vein comprises (a) forming a microchannel and a venousvalve positioned along the microchannel in a master mold, wherein thevenous valve comprises a pair of leaflets defining a pair of cusps ofthe venous valve, and a flow channel positioned between the leaflets,and (b) coating at least a portion of the microchannel with endothelialcells that form vascular lumen. In some embodiments, the endothelialcells comprise human umbilical vein endothelial cells (HUVECs) coatedover a layer of an extracellular matrix (ECM). In some embodiments, (b)comprises treating the vascular lumen with tumor necrosis-factor alpha(TNF-α) at a dosage of less than 300 nanograms per milliliter (ng/ml).

BRIEF DESCRIPTION OF THE DRAWINGS

For a detailed description of exemplary embodiments of the invention,reference will now be made to the accompanying drawings in which:

FIG. 1A is a schematic representation of a human vein;

FIG. 1B is a zoomed-in schematic representation of the human vein ofFIG. 1A having normal blood flow;

FIG. 1C is a zoomed-in schematic representation of the human vein ofFIG. 1A having DVT;

FIG. 1D is a zoomed-in schematic representation of the human vein ofFIG. 1A having an embolism;

FIG. 2 is a schematic representation of a venous valve;

FIGS. 3-5 are schematic representations of an embodiment of amicrofluidic chip in accordance with principles disclosed herein;

FIG. 6 is a graphical illustration of simulated fluid flow through anembodiment of a venous valve of the microfluidic chip of FIGS. 3-5 inaccordance with principles disclosed herein;

FIG. 7 is an image indicating the fully formed endothelium in themicrofluidic chip of FIGS. 3-5;

FIG. 8 is an image indicating platelet deposition in an embodiment of avenous valve of the microfluidic chip of FIG. 4 in accordance withprinciples disclosed herein;

FIG. 9 is an image indicating fibrin/fibrinogen deposition in anembodiment of a venous valve of the microfluidic chip of FIG. 4 inaccordance with principles disclosed herein;

FIG. 10 is another image indicating platelet deposition in the venousvalve of FIG. 8;

FIG. 11 is another image indicating fibrin/fibrinogen deposition in thevenous valve of FIG. 9;

FIGS. 12-30 are graphs illustrating experimental data pertaining toembodiments of venous valves of the microfluidic chip of FIG. 4 inaccordance with principles disclosed herein;

FIGS. 31-33 are Brightfield microscopic images of an embodiment of avenous valve of the microfluidic chip of FIG. 4 in accordance withprinciples disclosed herein;

FIG. 34 is a graph illustrating experimental data pertaining to thevenous valve shown in FIGS. 31-33;

FIG. 35 is a scanning electroscope micrograph of the venous valve shownin FIGS. 31-33;

FIGS. 36, 37 are images of an embodiment of a venous valve of themicrofluidic chip of FIG. 4 in accordance with principles disclosedherein;

FIG. 38 is a graph illustrating experimental data pertaining to thevenous valve shown in FIGS. 36, 37;

FIG. 39 is a perspective view of an embodiment of a macroscale actuatingvenous valve model in accordance with principles disclosed herein;

FIG. 40 is a cross-sectional view of the actuating venous valve model ofFIG. 39 along line 40-40 of FIG. 39;

FIG. 41 is a cross-sectional view of the venous valve model of FIG. 39along line 41-41 of FIG. 40;

FIG. 42 is a cross-sectional view of the venous valve model of FIG. 39along line 42-42 of FIG. 40;

FIG. 43 is a bottom view of an embodiment of a central layer of thevenous valve model of FIG. 39 in accordance with principles disclosedherein;

FIG. 44 is a bottom view of an embodiment of a venous valve of thecentral layer of FIG. 43 in accordance with principles disclosed herein;and

FIG. 45 is a cross-sectional view of the venous valve model of FIG. 39along line 45-45 of FIG. 40.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following discussion is directed to various exemplary embodiments.However, one skilled in the art will understand that the examplesdisclosed herein have broad application, and that the discussion of anyembodiment is meant only to be exemplary of that embodiment, and notintended to suggest that the scope of the disclosure, including theclaims, is limited to that embodiment.

Certain terms are used throughout the following description and claimsto refer to particular features or components. As one skilled in the artwill appreciate, different persons may refer to the same feature orcomponent by different names. This document does not intend todistinguish between components or features that differ in name but notfunction. The drawing figures are not necessarily to scale. Certainfeatures and components herein may be shown exaggerated in scale or insomewhat schematic form and some details of conventional elements maynot be shown in interest of clarity and conciseness.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

In the following discussion and in the claims, the terms “including” and“comprising” are used in an open-ended fashion, and thus should beinterpreted to mean “including, but not limited to . . . .” Also, theterm “couple” or “couples” is intended to mean either an indirect ordirect connection. Thus, if a first device couples to a second device,that connection may be through a direct engagement between the twodevices, or through an indirect connection that is established via otherdevices, components, nodes, and connections. In addition, as usedherein, the terms “axial” and “axially” generally mean along or parallelto a particular axis (e.g., central axis of a body or a port), while theterms “radial” and “radially” generally mean perpendicular to aparticular axis. For instance, an axial distance refers to a distancemeasured along or parallel to the axis, and a radial distance means adistance measured perpendicular to the axis. Any reference to up or downin the description and the claims is made for purposes of clarity, with“up”, “upper”, “upwardly”, “uphole”, or “upstream” meaning toward thesurface of the borehole and with “down”, “lower”, “downwardly”,“downhole”, or “downstream” meaning toward the terminal end of theborehole, regardless of the borehole orientation. As used herein, theterms “approximately,” “about,” “substantially,” and the like meanwithin 10% (i.e., plus or minus 10%) of the recited value. Thus, forexample, a recited angle of “about 80 degrees” refers to an angleranging from 72 degrees to 88 degrees.

Referring to FIGS. 1A-2, as described above, human veins (e.g., humanvein 10 shown in FIG. 1A) may, from a condition of normal blood flow 12(shown schematically in FIG. 1B), develop DVT 14 (shown schematically inFIG. 1C) which may result in potentially fatal venous thromboembolism 16(shown schematically in FIG. 1D). Ex vivo and in vivo animal studies aregenerally expensive, time-consuming, and often fail to model biologicalresponses in humans. The present disclosure is directed towardsleveraging microengineering technology known as “organ-on-a-chip” forcontrolling cellular microenvironments with high spatiotemporalprecision, and to present living cultured cells with mechanical andbiochemical signals in a more physiologically relevant context. Bydeploying this strategy, in vitro models of diseases of the human veinsmay be developed that include a design and biological complexity similarto that of human veins. The organ-on-a-chip methodologies disclosedherein offer a more physiologically-relevant human disease modelingplatform for DVT 10. Particularly, a micro-physiological DVT-on-a-chipis disclosed herein that is a mouse-scale model of a vein containing avenous valve 20 (represented schematically in FIG. 2) comprising a valveleaflet 22 and having a sinus depth 24, a sinus width 26, a gap width28, and a channel width 30. The DVT-on-a-chip disclosed herein allowsfor the independent or cooperative perturbing of Virchow'striad-endothelial valve architecture and state of activation (includinghypoxia), whole blood flow, and blood cells and coagulationfactors/proteins to thereby permit dissectible analysis of DVT.

Referring to FIGS. 1A-5, an embodiment of a vein-on-a-chip, venous valvemodel, or microfluidic chip 40 is shown schematically in FIGS. 3-5.Microfluidic chip 40 comprises a chip or body 42 through which threeseparate fluid microchannels 44A, 44B, and 44C linearly extend(microchannels 44A-44C are shown separately in FIGS. 3-5, respectively).Each microchannel 44A, 44B, 44C includes a fluid inlet, a fluid outlet,and a plurality of venous valve 50A, 50B, and 50C, respectively.

Particularly, in the embodiment of FIGS. 3-5, first fluid channel 44Aincludes three first venous valves 50A (only one of which is shown inFIG. 3), second fluid channel 44B includes three second venous valves50B (only one of which is shown in FIG. 4), and third fluid channel 44Cincludes three third venous valves 50C (only one of which is shown inFIG. 5). Each venous valve 50A-50B comprises a pair of valve leaflets 51defining cusps 52, and a central flow channel 54 extending between thepair of leaflets 51.

In this embodiment, each first venous valve 50A is 25% open (e.g.,having a gap width across flow channel 54 of about 50 micrometers (μm)),each second venous valve 50B is 50% open (e.g., having a gap width ofabout 200 μm), and each third venous valve 50C is 75% open (e.g., havinga gap width of about 150 μm). In this embodiment, body 42 ofmicrofluidic chip 40 is about 75 millimeters (mm) long and 25 mm wide.Additionally, in this embodiment, each microchannel 44A-44C is about twocentimeters (cm) long, about 200 μm wide, and about 75 μm in height,which may be similar in size and geometry as a mouse vein. Further, inthis embodiment, each venous valve 50A-50C spaced about 0.5 cm apartalong microchannels 44A-44C, respectively. In some embodiments, body 12is formed from polydimethylsiloxane (PDMS), a silicon-based organicpolymer, via soft lithography. One or more pumps may be coupled tomicrochannels 44A-44C for flowing or directing fluid through eachmicrochannel 44A-44C. Additionally, a microscope may be positioned overeach venous valve 50A-50C of each microchannel 44A-44C, respectively,for monitoring fluid flow through venous valves 50A-50C.

In this embodiment, to form microfluidic chip 40, microelectronic andsemiconductor fabrication techniques were used to develop an SU-8photoresist-based master mold. Particularly, computational fluiddynamics simulations were carried out to arrive at the open venous valvedesigns (e.g., venous valves 50A-50C) which contained recirculations asobserved in venous valve cusps in vivo. The embodiments of the finalizedvenous valve designs shown in FIGS. 3-5 were developed using solidmodeling computer-aided design (CAD) software (e.g., the SolidWorks™software package published by Dassault Systèmes) and a photomask of thefinalized valve designs was used to fabricate the master mold usingepoxy-based negative photoresist—SU-8 on a silicon wafer in thisembodiment. In this embodiment for forming microchannels 44A-44C, oncethe master molds were fabricated, the wafers were treated with Silane,and Polydimethylsiloxane (PDMS) having an epoxy to crosslinker ratio ofabout 10:1 was poured into the master mold. In this embodiment, once thePDMS was set, the PDMS was striped and bonded to a PDMS coated glassslide to form microchannels 44A-44C, each microchannel 44A-44C havingPDMS coated on each side thereof. Additionally, PDMS, being hydrophobic,was treated with plasma and then silanized with (3-aminopropyl)triethoxylsilane (APTES) to make the PDMS hydrophilic. In thisembodiment, microchannels 44A-44C were then filled with type-I rat tailcollagen of about 200 micrograms per milliliter (ug/ml) and fibronectinof about 50 ug/ml to form an extracellular matrix (ECM) configured tosupport endothelial cell growth on the walls of each microchannel44A-44C.

In this embodiment for forming microchannels 44A-44C, human umbilicalvein endothelial cells (HUVECs) were cultured over a layer of the ECMcoated on microchannels 44A-44C; however, in other embodiments, human oranimal cell lines other than HUVECs may also be used in the formation ofmicrochannels 44A-44C. In this embodiment, HUVECs, a human primary cellline derived from the human umbilical vein, were acquired and seeded onT75 flasks coated with type I rat tail collagen of about 5 micrograms(μg) per 5 cm², the HUVECs being seeded at about 50,000 cells per flask.In this embodiment, the endothelial growth media (EGM) (EGM-2 MV,promocell in this embodiment) disposed in the flasks were replaced everytwo days, and once the flasks were 80% confluent, the HUVECs weredetached from the flasks and seeded on microchannels 44A-44C with about20-30 microliters (μl) of cell suspension having cell density of about1e7 cells per milliliter (ml). The cell suspension was passed through acell strainer to remove debris and cell aggregates that were larger thanabout 40 μm in diameter. In this embodiment, microchannels 44A-44C werethen first filled with cell suspension and kept upside down for about 20minutes to seed the top face of microchannels 44A-44C, and thenmicrochannels 44A-44C were again filled with cell suspension and keptupright for 20 minutes to seed the bottom faces of microchannels44A-44C.

To induce activation, the vascular lumen coated to microchannels 44A-44Cwas treated with the tumor necrosis factor (TNF) TNF-α at about 0-200nanograms per milliliter (ng/ml) in this embodiment. Particularly, theseeded microchannels 44A-44C were perfused with media at about 1 ul/minfor about 24 hours. Confluent lumen was formed on the walls ofmicrochannels 44A-44C at the end of 24 hours. Additionally, in thisembodiment, different doses TNF-α, a cytokine, was introduced into theconfluent lumen of the channels for about 18 hours to observe the dosedependent inflammation of the endothelial cells in the straight sectionsof microchannels 44A-44C and venous valve cusps of venous valves50A-50C.

Further, in this embodiment of the formation of microchannels 44A-44C,computation fluid dynamics (CFD) simulations of non-Newtonian blood flowwere carried out via software (e.g., ANSYS® software published by AnsysInc.) to predict disturbed venous blood flow. Particularly, referring toFIGS. 1A-206, CFD simulations were performed for the venous valves50A-50C of microchannels 44A-44C to analyze the characteristics ofnon-Newtonian fluid flow therethrough, as shown particularly in FIG.206. For example, a CFD simulation 60 was performed for second venousvalve 50B. The CFD simulation 60 confirmed the formation of secondaryvortices and stasis of blood in the valve cusps 52 of the simulatedvenous valve 50B′ (shown in FIG. 6), predicting the tendency of thegeometries of simulated venous valve 50B′ to form thrombus within valvecusps 52.

In this embodiment of the formation of microchannels 44A-44C, toevaluate role of hypoxia, microfluidic chip 40 was incubated at 3.9%oxygen. Re-calcified citrated blood was perfused through microchannels44A-44C at a physiological or pathological shear stress (about 0.5-20dynes per centimeters squared (dynes/cm²) in this embodiment).Additionally, blood coagulation in microchannels 44A-44C was altered bythe addition of thrombin or heparin. Fluorescently labelled plateletsand fibrin were visualized and quantitated via microscopes positionedadjacent microchannels 44A-44C. Additionally, in this embodiment,typical vascular identity and adhesion markers were measured throughimmunohistochemistry.

Referring to FIGS. 3-5 and 7-11, exemplary imagery pertaining toexperimental whole blood (tagged for platelets and fibrin/fibrinogen)perfusion through embodiments of venous valves 50A-50C is shown in FIGS.7-11. As shown particularly in image 62 of third venous valve 50Cpresented in FIG. 7, confluent vascular lumens 64 were cultured withinvenous valves 50A-50C with expected junction integrity, actin andvisible nuclei. In this example, when blood was perfused through theuntreated healthy HUVECs shown in venous valves 50A-50C at venous shearof about 0.5 dynes/cm², no thrombi were detected. For example, image 65of FIG. 8 illustrates platelets within venous valve 50B while image 66of FIG. 9 illustrates fibrinogen/fibrin within venous valve 50B.However, upon stimulation of HUVECs with TNF-α (shown in FIGS. 10, 11),thrombi were detected due to the expression of endothelial adhesionproteins. For example, image 67 of FIG. 10 illustrates platelets withinvenous valve 50B while image 68 FIG. 11 illustrates fibrogen/fibrinwithin venous valve 50B.

Referring to FIGS. 3-5, 12-30, experimental data pertaining to exemplaryperfusion of blood through venous valves 50A-50C is shown in FIGS.12-30. Particularly, graphs 70, 71 shown in FIGS. 12, 13, respectively,illustrate TNF-α dosage in venous valve 50B and fibrin in cusps 52 ofvenous valves 50A-50C, while graphs 72, 73 of FIGS. 14, 15,respectively, illustrate platelet distribution and fibrin formation invenous valve 50B. Additionally, graphs 74, 75 shown in FIGS. 16, 17,respectively, illustrate platelet deposition at high shear (about 15dyne/cm in the exemplary experimental data provided in FIGS. 12-30) andvenous shear (about 1 dyne/cm in the exemplary experimental dataprovided in FIGS. 12-30) and fibrin formation at high shear versusvenous shear in venous valve 50B, while graphs 76, 77 shown in FIGS. 18,19, respectively, illustrate platelet deposition and fibrin formationfor each venous valve 50A, 50B, and 50C. Further, graphs 78, 79 shown inFIGS. 20, 21, respectively, illustrate platelet (ECM) distribution andfibrin formation versus lumen (HUVEC) deposition in venous valve 50B.

Graph 80 shown in FIG. 22 illustrates both platelet and fibrin coveragewithin cusps 52 of venous valve 50B for high shear versus venous shear,while graph 81 shown in FIG. 23 illustrates fibrin formation for TNF-αdosages of 0 ng/ml, 5 ng/ml, and 200 ng/ml in venous valve 50B. Graph 82shown in FIG. 24 illustrates fibrin formation within cusps 52 of venousvalve 50B for TNF-α dosages of 0 ng/ml, 5 ng/ml, and 200 ng/ml. Graph 83shown in FIG. 25 illustrates intercellular adhesion module (ICAM)-Iexpression for TNF-α dosages of 0 ng/ml, 200 ng/ml in venous valve 50Bwhile graph 84 shown in FIG. 26 illustrates ICAM-1 expression in thecusps 52 of venous valves 50B for TNF-α dosages of 0 ng/ml, 200 ng/ml.Additionally, graph 85 shown in FIG. 27 illustrates platelet depositionwithin venous valve 50B for TNF-α dosages of 0 ng/ml, 5 ng/ml, and 200ng/ml, while graph 86 shown in FIG. 28 illustrates fibrin formationwithin the cusps 52 (at a TNF-α dosage of 5 ng/ml) of each venous valve50A-50C. Further, graph 87 shown in FIG. 29 illustrates expression ofVon Willebrand factor (vWF) within venous valve 50B for TNF-α dosages of0 ng/ml and 200 ng/ml, while graph 88 shown in FIG. 30 illustrates vWFexpression within the cusps 52 of venous valve 50B for TNF-α dosages of0 ng/ml and 200 ng/ml.

In this example, increased fibrin deposition occurred at the cusps 52 ofvenous valves 50A-50C and very limited platelet deposition alsooccurred, which is typical for venous thrombi in vivo. Also in thisexample, the flow rate of blood flow was varied through microchannels44A-44C and it was found that thrombi formed in the cusps 52 are flowdependent. At venous shear stress, fibrin-rich thrombi at the cusps wereobserved in this example whereas platelet adhesion was observed only athigh shear. Further, in this example, addition of heparin in blooddecreased the thrombi formation within venous valves 50A-50C which wasdose dependent.

In view of the above, including the experimental data illustrated inFIGS. 12-30, microfluidic chip 40 provides for the modelling anddissection of critical biophysical and biological processes of DVTunobserved in straight perfusion devices or conventional mouse models.Additionally, microfluidic chip 40 may be used to help unravel specificshear and endothelium driven signaling pathways, and drug-tissueinteractions.

Referring to FIGS. 31-35, Brightfield microscopic images 101, 102 of acusp 52 of an embodiment of venous valve 50B are shown in FIGS. 31-33.Particularly, image 101 shown in FIGS. 31, 32 (FIG. 32 being a zoomed-inview of image 101 shown in FIG. 31, where image 101 in FIG. 31 has ascale of approximately 200 μm in this example) illustrate cusp 52 at afirst moment in time while image 103 shown in FIG. 33 (FIG. 33 havingthe same scale as FIG. 32, approximately 50 μm in this example)illustrate cusp 52 at a second moment in time approximately 0.18 secondsafter the first moment. As shown particularly in FIGS. 31-33, the fluidflow pattern, recirculations and secondary vortices were recreated usingan embodiment of microfluidic chip 40 similar to that observed in vivo.Particularly, images 101, 103 illustrate the formation of vortices(indicated by arrows 105 in FIG. 33) of red blood cells 107 within cusp52. In the example shown in FIGS. 31-35, red blood cells 107 follow acircular path in the primary vortex 105. FIG. 34 illustrates a graph 110comparing velocity within primary vortex 105 as estimated from theexperiment shown in FIGS. 31-33 and that projected from CFD analysis.

As shown particularly in FIG. 35, the constituents of thrombi formed incusp 52 may be analyzed from scanning electron microscope (SEM) images,such as the scanning electroscope micrograph or image 112 shown in FIG.35. In the example shown in FIG. 35, the formed thrombi were first fixedusing paraformaldehyde and then the embodiment of microfluidic chip 40used in this example was cut open along microfluidic channels 44A-44C.Once cut, microfluidic channels 44A-44C were sputter coated with a layerof gold nanoparticles and imaged in a SEM. Image 112 of thrombi(indicated by arrows 114 in FIG. 35) formed in cusp 52 reinforces thatthe thrombi 114 are rich in fibrin/red blood cells and devoid ofplatelets.

Conventional treatment of DVT often includes the prescription ofanticoagulants, which alters blood chemistry. However, there is nogeneral clinical consensus upon the type and dosage of anticoagulantthat is best suited to treat DVT and often treatment with anticoagulantssignificantly increases the risk of bleeding in patients. Embodiments ofthe microfluidic chip 40 may be utilized to assess anticoagulationtherapy in DVT. Referring to FIGS. 36-38, images 120, 122 are shown ofembodiments of venous valve 50B (treated with TNF-α at a dosage of 5ng/ml) perfused with blood treated with Heparin lock flush, anintravenously administered drug that inactivates coagulation factors.Particularly, image 120 illustrates venous valve 50B perfused with bloodtreated with Heparin having a dosage of approximately 0.25 InternationalUnits per milliliter (IU/ml) while image 122 illustrates venous valve50B perfused with blood treated with Heparin having a dosage of 0.50IU/ml. Additionally, a graph 124 shown in FIG. 38 illustratesexperimental data derived from the perfusion of Heparin treated bloodthrough venous valve 50B as shown in FIGS. 36, 37.

In the example of FIGS. 33-38, thrombi formed in microfluidic channels44A-44C reduced significantly when the dosage of Heparin was increased,as shown in graph 124 of FIG. 38. However, while a reduction in fibrinresulted even at a lower dose of Heparin within microfluidic model 40,fibrin in cusps 52 only reduced at the higher Heparin dosage (0.50 IU/mlin this example). The data provided by graph 124 indicates that venouscusps (e.g., cusps 52) may require higher Heparin anticoagulation thanthe systemic heparin anticoagulation to completely prevent the formationof local thrombi.

As shown particularly in the graph 124 of FIG. 38, embodiments of venousvalve 50B were also perfused with blood treated by Rivaroxaban (atdosages of 100 ng/ml and 500 ng/ml), and blood treated with Apixaban (atdosages of 50 ng/ml and 500 ng/ml), each of which compriseclinically-prescribed direct oral anticoagulants (DOACs) that are potentantithrombotic drugs configured to inhibit factor Xa. In this example,when added to blood samples and introduced into an embodiment of venousvalve 50B treated with TNF-α, fibrin rich clots were present in cusps 52at the standard dosage for each DOAC (100 ng/ml of Rivaroxaban and 50ng/ml of Apixaban, respectively) while there were unobservable thrombiin the luminal portion of the microfluidic model 40. Further, only whenthe dosage of each DOAC was increased to 500 ng/ml were thrombi detectedat cusps 52 and at the microfluidic channel 44B. Taken together,microfluidic model 40 validated the role of blood plasma chemistry as aVirchow factor that regulates DVT and predicted that standard-of-careanticoagulant therapy may resolve vein thrombosis mostly when prescribedat high doses, thus making patients more vulnerable to bleeding.

The present disclosure is also directed towards a three-dimensionally(3D)-printed fabrication technique for creating a 3D-printed vein ormacroscale model of venous architecture having the same or similardimensions as an in vivo human vein (e.g., the length of a side of theprinted vein equals, or is similar to, the diameter of an in vivo vein).Additionally, in some embodiments, the 3D printed vein is integratedwith mechanical and electrical instrumentation that can actuate,modulate, and predict the contractile (pumping) phenomena of the veinsas well as pulsatile blood flow through the macroscale model. Further,the 3D-printed vein may be applied for studying the blood rheology,flow, initiation of clots and anticoagulant dosage in DVT.

In some embodiments, a macroscale model of a human vein may be formedusing a CAD model (created via, e.g., the SolidWorks™ software package)having a square cross-section and comprising a scaled-up version ofmicrofluidic chip 40 matching the dimensions of a human vein. As will bedescribed further herein, the macroscale model may be provided withcompartments on either side of a central channel and fabricated by 3Dprinting. The compartments may incorporate dynamic actuation to mimicthe actuation and pumping of blood observed in human veins. Embodimentsof the macroscale model may comprise an approximately 12 cm long centralchannel having an approximately 6 mm by 6 mm square cross-section.Embodiments of the macroscale model may be 3D printed on, for example, aStratasys® Connex™ 500 multi-material printer using TangoPlus™ printingmaterial, or other similar instrumentation and materials.

Additionally, in an exemplary embodiment for forming a macroscale modelof a human vein, CFD simulations and nonlinear static structuralsimulations were conducted using CFD and finite element analysis (FEA)software, such as ANSYS® Fluent and ANSYS® Mechanical APDL,respectively, to determine the contour shape of the central channel ofthe macroscale model which when actuated compresses and simultaneouslyopens venous valves of the macroscale model. A CAD model of themacroscale model may be built using a CAD software package (e.g., theSolidWorks™ software package) and converted into a 3D-printablestereolithography (STL) format. Embodiments of the macroscale model maybe fabricated with VeroWhite™ and TangoPlus™ printing materials.Particularly, embodiments of the macroscale model comprise a centralchannel having side walls printed with VeroWhite™, and top and bottomfaces printed with TangoPlus™.

Referring to FIGS. 39-45, an embodiment of a 3D printed macroscale, invivo human venous valve model 200 is shown. Venous valve model 200 has acentral or longitudinal axis 205 and generally includes a body 202including an upper layer 204 (shown particularly in FIG. 41), a centrallayer 20210 (shown particularly in FIGS. 42-44), and a lower layer 220(shown particularly in FIG. 45). Upper layer 204 of venous valve modelincludes an upper air channel 206 having a generally squarecross-section, central layer 20210 includes a central fluid channel 212having a generally square cross-section and which extends adjacent theupper air channel 206 of upper layer 204, and lower layer 220 includes alower air channel 222 having a generally square cross-section whichextends adjacent the fluid channel 212 of central layer 210.

In this embodiment, upper air channel 206 and lower air channel 222 areeach in fluid communication with an air inlet 207 and an air outlet 222of venous valve model 200, where air inlet 207 and air outlet 209 eachextend orthogonal central axis 205. In some embodiments, air inlet 207may be in communication with a pressure regulator (not shown in FIGS.39-45). to provide a predetermined air pressure within channels 207,209. In some embodiments, an air pressure of approximately between eightand 11 kilopascals (kPa) may be provided in air channels 206, 222. Airchannels 206, 222 extend parallel with fluid channel 212 and, as shownparticularly in FIG. 40, air channels 206, 222 each lie within the samevertical plane 215 (plane 215 extending orthogonal central axis 205) asfluid channel 212. Also as shown particularly in FIG. 40, upper airchannel 206 extends entirely through a bottom face 208 of upper layer204, and thus air within upper air channel 206 contacts and actsdirectly against an upper wall 226 of fluid channel 212. Additionally,in this embodiment, fluid channel 212 extends through a bottom face 217of central layer 210, and thus fluid within fluid channel 212 contactsand acts directly against an upper wall 228 of lower air channel 222. Insome embodiments, walls 226, 228 may each be approximately 20 μm thick.

In the embodiment of FIGS. 39-45, fluid channel 212 of central layer 220extends parallel with central axis 205 and extends from a fluid inlet213 and a fluid outlet 215 opposite fluid outlet 213. In someembodiments, fluid channel 212 may be aligned with central axis 205whereby a plane extends through both fluid channel 212 and central axis205. Fluid channel 212 includes three actuatable venous valves 214, eachof which may be selectably opened and closed to control the flow offluid through fluid channel 212. Additionally, central layer 20210includes a plurality of actuation channels or compartments 216.

In vivo deep human veins are generally actuated by the surroundingmuscles in which the vein lies. In this embodiment, the actuation ofvenous valve model 200 is achieved by the actuation of actuationcompartments 216 that are separated from fluid channel 212 by walls 218that are approximately 250 μm in thickness. Actuation compartments 216on either side of fluid channel 212 are further divided into four partsby thin separating walls 219 approximately 250 μm in thickness.Separating walls 219 are placed near venous valves 214 such that eachactuation compartment 216 is partially defined by one of the walls 218of fluid channel 212 and a pair of separating walls 219, where wall 218of fluid channel 212 extends between the pair of separating walls 219and adjacently positioned venous valves 214. In this embodiment, fluidchannel 212 has a length 212L extending from fluid inlet 213 to fluidoutlet 213 that is approximately 2 cm, and a width 212W extendingbetween walls 218 that is approximately 200 μm.

In this embodiment, each actuation compartment 216 is in fluidcommunication with one of a plurality of pumps 230A-230D (shownschematically in FIG. 43) via a channel 232 extending orthogonal centralfluid channel 212. In some embodiments, each pump 230A-230D may comprisea syringe pump such as a Harvard apparatus; however, in otherembodiments, pumps 230A-230D may comprise other types of pumps,including commercially available vacuum pumps. In this embodiment, eachpump 230A-230D is connected to a corresponding channel 232 via tubing231 having an inner diameter of approximately 0.5 millimeter (mm). Inthis configuration, when one of the pumps 230A-230D is actuated in awithdraw mode, the actuated pump 230A-230D will dilate the portion ofthe fluid channel 212 located adjacent the actuation compartment 216 influid communication with the actuated pump 230A-230D by deforming thewall 218 separating the actuation compartment 216 and the fluid channel212. Similarly, when one of the pumps 230A-230D is actuated in aninfusion mode, the actuated pump 230A-230D will compress the portion offluid channel 212 located adjacent the actuation compartment 216 influid communication with the actuated pump 230A-230D.

In this embodiment, venous valve model 200 comprises three venous valves214 and four linear sections 212A-212D (shown in FIG. 43) of fluidchannel 212; however, in other embodiments, the number of venous valves214 and sections 212A-212D of fluid channel 212 may vary. When each wall218 of the same section 212A-212D (e.g., section 212B in this example)of fluid channel 212 are compressed, some of the fluid (blood or bloodmimicking fluids) inside section 212B of fluid channel 212 is displacedor pumped forward into section 212C (located immediately downstream fromsection 212B) via the actuation of pumps 230B into the infusion modewhile the remainder of the fluid in section 212B of fluid channel 212 isdisplaced or forced upstream in the direction of section 212A(positioned immediately upstream from section 212B of fluid channel212). The portion of the fluid displaced downstream flows through thevenous valve 214 positioned between sections 212B, 212C and into section212C of fluid channel 212. The portion of the fluid displaced upstreamfrom the compressed portion of fluid channel 212 flows into the cusps223 (shown in FIG. 44) of the venous valves 214 positioned betweensections 212A, 212B to trap and thereby restrict the fluid from flowinginto section 212A of fluid channel 212. Particularly, each venous valvecomprises a pair of valve leaflets 225 defining cusps 223, and a centralflow channel 227 extending between the pair of leaflets 225. Thus, cusps223 of venous valves 213 enable the unidirectional flow of fluid throughvenous valve model 200 as seen in vivo. Air channels 206 and 222 ofvenous valve model 200 permit deformation of fluid channel 212 tothereby assist with the flowing of fluid through fluid channel 212 viavenous valves 214.

In some embodiments, venous valves 214 are actuated (opened and closed)by contracting and dilating sections 212A-212D of fluid channel 212 inan alternating manner. For example, pumps 230A, 230C may be actuated inthe infusion mode to contract sections 212A, 212C of fluid channel 212while pumps 230B, 230D are actuated in the withdraw mode to dilatesections 212B, 212D of fluid channel 212. The alternating contractionand dilations assists with one way pumping of the fluid in fluid channel212. In this embodiment, a 3D printable fixture or pumping system 250(shown schematically in FIG. 43) may be used to modify the working ofone or more of pumps 230A-230D to achieve the alternating actuation offluid channel 212. For example, the pumping system 250 may accommodatetwo syringes within it, with the pumping system 250 acting to drive onesyringe opposite to the other, i.e. when one syringe is in withdraw modethe other will be in infusion mode and vice versa. One of the syringesmay be connected to the actuation compartments 216 corresponding tosections 212A, 212C of fluid channel 212 while the other syringe may beattached to the actuation compartments 216 corresponding to sections212B, 212 D of fluid channel 212. In other words, pumps 230A, 230C maycomprise a first syringe of the pumping system 250 while pumps 230B,230D may comprise a second syringe of the pumping system 250.

In this embodiment, endothelial cells were grown on the walls (includingupper wall 226) of the fluid channel 212 of an embodiment of venousvalve model 200 to form confluent lumen that mimics in-vivo blood vesselphysiology. Also, media was perfused in these channels for anapproximately twenty-four hour period at constant and pulsatile venousflow rates to induce wall shear stress similar to that experienced byendothelial cells in the in vivo deep veins. The cells were then treatedwith different doses of cytokines (e.g., 0 ng/ml, 5 ng/ml and 100 ng/mlfor about eighteen hours) to recapitulate the physiology of diseased andhealthy venous valves before perfusing blood derived from healthyindividuals and diseased patients.

Conventional techniques for modeling human veins generally comprise theuse of animal models which do not mimic the anatomy, physiology andbiophysics of venous architecture and flow. Therefore, attempting drugdiscovery with these models is not predictive. The microfluidic andmacroscale models (e.g., microfluidic model 40 and venous valve model200) described herein may address some of the limitations ofconventional modeling techniques. For example, the microfluidic andmacroscale models described herein may be made in vitro with materialsand techniques that permit convenient analysis of the biology of venousflow and vessel using microscopy and other biochemical assays.Additionally, the in vitro microphysiological model (e.g., microfluidicchip 40) of deep veins may recapitulate the in vivo microphysiologyfaithfully. As described above, the fluid flow pattern, recirculationsand secondary vortices were recreated in embodiments of microfluidicmodel 40 similar to that observed in vivo. As low as 20 ul of blood wasenough to carry out a single experiment with embodiments ofmicrochannels 44A-44C as channels each only have a volume ofapproximately 0.3 ul. Further, a bioprinted macroscale model of a humanvein (e.g., venous valve model 200) was created with functioning,actuatable valves. The compression of the channel walls (e.g., walls 218of fluid channel 212) led to the opening of the venous valves (e.g.,valves 214) and expansion of the channel walls led to the closing of thevenous valves thus mimicking the actuation mechanism found in vivo. Themacroscale model was also able to include mechanical stimulus on top offlow induced shear stress to the cells if cells are cultured in thesechannels.

DVT and subsequent pulmonary embolism (PE) causes about 200,000 deathsin the US annually. The models and processes described herein may beused to develop a clinical device which will give a patient specificreadout for propensity for thrombogenesis. The models described hereinmay also be used in preclinical trials of new anticoagulant drugs inplace of animal models and human volunteers. The pharmaceutical industrymay use the models described herein for identifying mechanisms of actionof drugs, compounds and molecules that may have therapeutic or toxiceffects on human body.

Conventional models used in preclinical trials of new anticoagulantdrugs often comprise human volunteers and in vivo animal models. Forexample, the most common animal model of thrombosis is the murine modelin which venous thrombosis is induced slowly by stasis or stenosis (bothby ligation) or rapidly by an acute injury (using free-radicals) of theinferior vena cava. Though these diverse mouse models have contributedimmensely in decoding several key mechanisms that govern thrombosis,capturing the thrombus dynamics in human-relevant conditions, as well aseffective in studying the role of genetic variation and differentclotting factors in thrombus formation, the physiological and geneticdifferences of these models with respect to humans limits themconsiderably, as evidenced by the fact several drug trials thatsucceeded in such animal models have failed in human clinical trials,thus contributing to high healthcare costs.

In addition, there is inherent risk of bleeding to human volunteersduring their participation in the preclinical trials for testing newanticoagulant drugs. Further, a widely used tool to assess and determineif a patient has to receive thromboprohylaxis is to use risk scores,such as Khorana, PROTECHT, Vienna CATS, CONKO etc. The Khorana score,for example, gives a score based on the patient's cancer type, BMI,leukocyte count, platelet count and hemoglobin level, and if the scoreis above three, the patient has a high propensity to get DVT. One of themain limitations with these scores is their predictive performance maybe limited and does not include the factors that enhances thrombusformation like coagulation factors.

The models and processes disclosed herein (e.g., microfluidic model 40and venous valve model 200) create human physiology outside of humanbody to test new anticoagulant drugs. Thus, use of animals and humanvolunteers in the preclinical and clinical trials may be reduced. Themodels disclosed herein may also be used for thromboprophylaxis andanticoagulant drug dosage from a readout by perfusing patient derivedblood. While conventional parallel plate flow chambers have been usefulin studying the effects of shear and recirculating flow on plateletfunction and coagulation, accurate blood vessel anatomy and flowpatterns are not replicated in these conventional devices. Modelsdisclosed herein however include accurate blood vessel anatomy and thecomplex flow pattern observed in vivo that are relevant to DVTformation. Conversely, conventional cone-and-plate viscometers are oftenbulky and need large amounts of blood, cultured cells and reagents foreach experiment, making them low throughput. Also, the experiments inthese conventional devices are typically conducted over two-dimensional(2D) monolayers of proteins or cells and therefore, they do not mimicthe function of a 3D round vascular lumen and natural blood flow. Atleast some of these limitations are addressed in the models disclosedherein. For example, embodiments of microfluidic model 40 only requiresa few microliters of blood and reagents are needed for each experimentand only about 10 million cells are needed to form a 3D confluent lumenof endothelial cells in an approximately twenty-four hour period. Themacroscale model (e.g., venous valve model 200) also accuratelysimulates the mechanical strains that are experienced by the endothelialcells in vivo.

Extensive simulations of the fluid flow in the microfluidic channels(e.g., microchannels 44A-44C) have been carried out and the resultssupport the formation of disturbed flow and vortices in the venous valvedesign. Endothelial cells were cultured in these channels and blood wasperfused at venous shear rates which resulted in free flow of bloodwithout any clot formation. This suggests that an intact lumen wasformed in the channel as observed in vivo. When the lumen was treatedwith varying doses of cytokines (TNF-α), fibrin rich clots werepredominantly formed in the venous valve cusps as observed in vivo.Additionally, the simulations of flow and actuation of the macroscopicmodel (e.g., venous valve model 200) using computational fluid dynamicsand structural assessment software tools have given positive results.

While exemplary embodiments have been shown and described, modificationsthereof can be made by one skilled in the art without departing from thescope or teachings herein. The embodiments described herein areexemplary only and are not limiting. Many variations and modificationsof the systems, apparatus, and processes described herein are possibleand are within the scope of the disclosure. For example, the relativedimensions of various parts, the materials from which the various partsare made, and other parameters can be varied. Accordingly, the scope ofprotection is not limited to the embodiments described herein, but isonly limited by the claims that follow, the scope of which shall includeall equivalents of the subject matter of the claims. Unless expresslystated otherwise, the steps in a method claim may be performed in anyorder. The recitation of identifiers such as (a), (b), (c) or (1), (2),(3) before steps in a method claim are not intended to and do notspecify a particular order to the steps, but rather are used to simplifysubsequent reference to such steps.

What is claimed is:
 1. A venous valve model, comprising: a first layerhaving a central axis and a fluid channel extending between a fluidinlet and a fluid outlet formed in the first layer, wherein the fluidchannel is defined by a pair of channel walls; a first venous valveformed in the first layer and positioned along the fluid channel; and apair of first actuation chambers positioned adjacent the channel wallsof the fluid channel, wherein the pair of first actuation chambers areconfigured to decrease a width of the fluid channel in response topressurization of the pair of first actuation chambers.
 2. The venousvalve model of claim 1, wherein the first layer is formed from athree-dimensionally printed material.
 3. The venous valve model of claim1, further comprising a pump in fluid communication with at least one ofthe pair of first actuation chambers, wherein the pump comprises aninfusion mode configured to increase a pressure within the at least oneof the pair of first actuation chambers to decrease the width of thefluid channel, and a withdraw mode configured to decrease a pressurewithin the at least one of the pair of first actuation chambers toincrease the width of the fluid channel.
 4. The venous valve model ofclaim 3, wherein the pump comprises a syringe pump.
 5. The venous valvemodel of claim 1, wherein the first venous valve comprises a pair ofleaflets defining a pair of cusps of the first venous valve, and a flowchannel positioned between the leaflets.
 6. The venous valve model ofclaim 5, wherein the pair of first actuation chambers are configured todecrease a width of the flow channel of the first venous valve inresponse to the pressurization of the pair of first actuation chambers.7. The venous valve model of claim 6, wherein: the pair of firstactuation chambers are positioned adjacent a first section of the fluidchannel; the first layer further comprises a pair of second actuationchambers positioned adjacent a second section of the fluid channellocated between the first section and the fluid outlet, and wherein thefirst venous valve is positioned between the first section and thesecond section; and the leaflets of the first venous valve areconfigured to direct fluid within the second section of the fluidchannel into the cusps of the first venous valve in response topressurization of the pair of second actuation chambers.
 8. The venousvalve model of claim 7, further comprising: a pair of third actuationchambers positioned adjacent a third section of the fluid channellocated between the second section and the fluid outlet; a second venousvalve positioned between the second section and the third section; and apumping system comprising a plurality of pumps and configured tosimultaneously pressurize the first section and the third section of thefluid channel and depressurize the second section of the fluid channel.9. The venous valve model of claim 6, wherein the first layer comprisesa pair of chamber walls positioned between the first pair of actuationchambers and the second pair of actuation chambers, wherein the pair ofchamber walls restrict fluid communication between the first pair ofactuation chambers and the second pair of actuation chambers.
 10. Thevenous valve model of claim 1, further comprising: a second layercomprising a first air channel extending parallel with the fluid channelof the first layer; and a third layer comprising a second air channelextending parallel with the fluid channel of the first layer, whereinthe first air channel, the second air channel, and the fluid channel areeach intersected by a plane extending orthogonally from the centralaxis.
 11. A microfluidic chip for modelling flow through a vein,comprising: a body comprising a microchannel extending between a fluidinlet and a fluid outlet, wherein at least a portion of the microchannelis coated with endothelial cells that form vascular lumen; and a venousvalve formed in the body and positioned along the microchannel, whereinthe venous valve comprises a pair of leaflets defining a pair of cuspsof the venous valve, and a flow channel positioned between the leaflets.12. The microfluidic chip of claim 11, wherein the endothelial cellscomprise human umbilical vein endothelial cells (HUVECs).
 13. Themicrofluidic chip of claim 12, wherein the HUVECs are coated over alayer of an extracellular matrix (ECM).
 14. The microfluidic chip ofclaim 11, wherein the vascular lumen is treated with tumornecrosis-factor alpha (TNF-α) at a dosage of less than 300 nanograms permilliliter (ng/ml).
 15. The microfluidic chip of claim 11, wherein atleast a portion of the pair of cusps is coated with the endothelialcells that form the vascular lumen.
 16. The microfluidic chip of claimB1, wherein a width of the flow channel of the venous valve is between25 micrometers (μm) and 200 μm.
 17. The microfluidic chap of claim 11,wherein the body is formed from Polydimethylsiloxane (PDMS).
 18. Amethod of forming a microfluidic chip for modelling flow through a vein,comprising: (a) forming a microchannel and a venous valve positionedalong the microchannel in a master mold, wherein the venous valvecomprises a pair of leaflets defining a pair of cusps of the venousvalve, and a flow channel positioned between the leaflets; and (b)coating at least a portion of the microchannel with endothelial cellsthat form vascular lumen.
 19. The method of claim 18, wherein theendothelial cells comprise human umbilical vein endothelial cells(HUVECs) coated over a layer of an extracellular matrix (ECM).
 20. Themethod of claim 18, wherein (b) comprises treating the vascular lumenwith tumor necrosis-factor alpha (TNF-α) at a dosage of less than 300nanograms per milliliter (ng/ml).